The effect of sGP on immune response when antigen exposure mimics natural infection.
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Abstract
<p>(A) Immunization study design. Female BALB/C mice were immunized IM with 50 µg of total DNA per immunization according to the schedule shown. Mice were immunized with a 3∶1 ratio of sGP Edit∶GP<sub>1,2</sub> Edit in pCAGGS. Control groups were immunized with sGP Edit or GP<sub>1,2</sub> Edit alone plus empty pCAGGS vector to keep total amount of immunizing DNA constant. (B) Comparison of antibody response against GP<sub>1,2</sub>. Mouse sera collected at week 6 were analyzed for anti-GP<sub>1,2</sub> antibodies by ELISA using GP<sub>1,2</sub> as coating antigen. (C) sGP competition ELISA. The ability of sGP to compete for anti-GP antibodies was determined by competition ELISA as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003065#ppat-1003065-g003" target="_blank">Figure 3B</a>. Pooled antisera were analyzed from mice immunized with a GP<sub>1,2</sub> Edit (blue), sGP Edit (red), or a 3∶1 ratio of sGP Edit∶GP<sub>1,2</sub>Edit (purple), and were diluted to give roughly equivalent anti-GP<sub>1,2</sub> signal. Competition ELISA was performed from antisera collected at both week 6 (light color) and week 12 (dark color) according to the immunization schedule. (D) Competition immunoprecipitation. Pooled antisera from sGPEdit+GP<sub>1,2</sub>Edit-immunized mice were incubated with no GP, purified sGP or GP<sub>1,2</sub> alone, or with fixed GP<sub>1,2</sub> and increasing concentrations of sGP to compete for anti-GP<sub>1,2</sub> antibodies. GP<sub>1,2</sub> was incubated with recombinant HA as a negative control, and precipitated and analyzed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003065#ppat-1003065-g003" target="_blank">Figure 3E,F</a>. (E) Neutralization of EBOV GP pseudovirus. Neutralizing activity of antisera was determined by incubating 500 pfu of GP<sub>1,2</sub>-pseudotyped virus with dilutions of pooled sGP+GP<sub>1,2</sub>-immunized (red), or empty pCAGGS vector-immunized (black) antisera. Neutralization was measured as decrease in luciferase expression compared to virus-only controls. (F) Interference of EBOV GP pseudovirus neutralization by sGP. The ability of sGP to interfere with antibody-dependent neutralization was determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003065#ppat-1003065-g004" target="_blank">Figure 4B</a>. Pooled sGP+GP<sub>1,2</sub>-immunized antisera were fixed at the dilution corresponding to 80% neutralization. Antisera were co-incubated with increasing dilutions of purified sGP (red) or purified influenza PR8 HA (blue), and rescue of infectivity was measured as described in methods.</p
